Knowledge of how facultative pathogens adapt to changing conditions when passing from an environmental reservoir into a human host is limited. Advances in our understanding of the basic mechanisms of adaptation to the host and induction of virulence genes will provide new molecular targets for therapy to reduce the large medical burden imposed by this diverse group of pathogens. In the water-borne intestinal pathogen Vibrio cholerae we have demonstrated that signaling by external amino acids modulates the cytoplasmic concentration of the secondary messenger cyclic diguanylate (c-diGMP), leading to reciprocal regulation of genes important for biofilm formation and virulence. We hypothesize that c-diGMP also mediates the transition from environmental to virulence gene expression upon entry into the host. In this project we will investigate the mechanism and general importance of c-diGMP control of virulence gene regulation. We will characterize the sensory, regulatory and enzymatic properties of a phosphorelay system that lowers the cellular c-diGMP concentration by hydrolysis of c-diGMP and is required for virulence. We will also characterize other c-diGMP hydrolytic proteins that exert control over the cellular c-diGMP concentration during the transition from environment to host. Finally, we will investigate the mechanism by which c-diGMP influences virulence gene transcription. We anticipate that this work will provide a working model of the central regulatory pathway in V. cholerae that initiates virulence gene expression upon entry into the host. We also anticipate that these studies will have application to a broad range of facultative prokaryotic pathogens since most appear to contain multiple c-diGMP synthetic and hydrolytic proteins. [unreadable] [unreadable]